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Chemical composition, antibacterial and

antioxidant activities of essential oil of
Eucalyptus globulus from Algeria

ARTICLE in INDUSTRIAL CROPS AND PRODUCTS · DECEMBER 2015

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 Industrial Crops and Products 78 (2015) 148–153

Contents lists available at ScienceDirect

Industrial Crops and Products

journal homepage: www.elsevier.com/locate/indcrop

Chemical composition, antibacterial and antioxidant activities of

essential oil of Eucalyptus globulus from Algeria
Lila Harkat-Madouri a,b , Boudria Asma a , Khodir Madani a , Zakia Bey-Ould Si Said a ,
Peggy Rigou c , Daniel Grenier d , Hanane Allalou a , Hocine Remini a , Abdennour Adjaoud a ,
Lila Boulekbache-Makhlouf a,∗
a
Laboratoire de Biomathématiques, Biophysique, Biochimie, et Scientométrie (L3BS), Faculté des Sciences de la Nature et de la Vie, Université de Bejaia,
06000 Bejaia, Algeria
b
Faculté des Sciences Biologiques, Université des Sciences et de la Technologie Houari Boumediene, Alger, Algeria
c
INRA, UMR 1083 Sciences pour l’oenologie, Montpellier, France
d
Groupe de Recherche en Écologie Buccale (GREB), Faculté de Médecine Dentaire, Université Laval, Québec, Québec, Canada

a r t i c l e i n f o a b s t r a c t

Article history: Essential oils are known for their use in various fields such as cosmetic, pharmaceutical and food indus-
Received 13 July 2015 tries. The aim of this work is to investigate the chemical composition of essential oils from Eucalyptus
Received in revised form 7 October 2015 globulus leaves (E. globulus) by gas-chromatography coupled with mass spectrometry (GC/MS) method,
Accepted 7 October 2015
and to evaluate their antioxidant capacity (DPPH radical scavenging effect, reducing power, and inhibi-
tion of lipid peroxidation activity) as well as their antibacterial activity, against periodontopathogenic and
Keywords:
cariogenic bacterial species, using microdilution method in 96-well microplates. In total, 26 compounds
Eucalyptus globulus
were identified with the predominance of oxygenated monoterpenes (78.58%); 1,8-Cineole (55.29%),
Essential oil
GC/MS analysis
Spathulenol (7.44%) and ␣-Terpineol (5.46%) being the main components. The analyzed oils exhibited a
Antibacterial activity weak antioxidant capacity, but a marked antibacterial activity against Gram negative bacteria, mainly for
Antioxidant capacity F. nucleatum ATCC 25586 (MIC = 1.14 mg/mL) and P. gingivalis ATCC33277 (MIC = 0.28 mg/mL). Therefore,
E. globulus essential oils may have a potential therapeutic application for the treatment of periodontal
diseases.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction antimicrobials (Bakkali et al., 2008). Due to the toxicological effect

Eucalyptus globulus belongs to the family of Myrtaceae which
is indigenous to Australia. It was introduced to Algeria in 1854
by Ramel (Boulekbache-Makhlouf et al., 2010), where it is now
widely distributed. Essential oils of E. globulus contain more than
20 compounds with a prevalence of 1,8-Cineole (Batish et al., 2008;
Boukhatem et al., 2014; Goldbeck et al., 2014; Maciel et al., 2010).
The limitation on the use of synthetic antioxidants and the
increase interest for natural non-toxic antioxidants has spawned
numerous studies on the antioxidant potential of essential oils.
Essential oils of plants are a mixture of various components such
as monoterpenes, sesqui-terpenes, alcohols, esters, aldehydes and
ketones, which are involved in the defense of the plant against
pests, herbivores, fungi, and bacteria (Batish et al., 2008). Fur-
thermore, essential oils and aromatic plants are known for their
multiple uses in flavor and fragrance, as preservatives, and as
∗ Corresponding author. Fax: +213 34 21 47 62.
E-mail address: lilaboulekbachemakhlouf@yahoo.fr (L. Boulekbache-Makhlouf).
of the synthetic products, renewed efforts were provided in respect
of the use of essential oils as natural antioxidants and preservatives
in the food processing, food supplement production and pharma-
ceutical industry (Wei and Shibamoto, 2007).
The essential oils of Eucalyptus species are widely used in the
world, the United States Food and Drug Authority considered them
as safe and non-toxic, even the Council of Europe has approved
the use of eucalyptus oils as flavoring agent in foods (Batish et al.,
2008). Consequently, a growing interest has been given to their use
in the scientific research field and industry as a natural food addi-
tive, drugs and cosmetics. (Goldbeck et al., 2014; Ishnava et al.,
2013). Several studies investigated the antioxidant potential of
essential oils from various Eucalyptus species such as E. polyan-
themos, E. perriniana, and E. camaldulensis (Barra et al., 2010; Lee
and Shibamoto, 2001; Singh et al., 2012). Singh et al. (2012) have
reported a strong antioxidant activity of decaying and fresh leaves
of E. tereticornis against DPPH, OH• and O2 radicals. However, Barra
et al. (2010) have reported a moderate DPPH scavenging activity
for oils extracted from aerial parts of E. camaldulensis and E. radi-
ate. Eucalyptus leaves are rich sources of essential oils, flavonoids

http://dx.doi.org/10.1016/j.indcrop.2015.10.015
0926-6690/© 2015 Elsevier B.V. All rights reserved.
 L. Harkat-Madouri et al. / Industrial Crops and Products 78 (2015) 148–153
or tanins, which are responsible for their antibacterial, larvicidal,
fumigant, antioxidant activities and anthelmintic properties. The
major compounds of E. globulus essential oils are 1,8-cineole (euca-
lyptol), aromadendrene, globulol, D-limonene and pinene, their
content depends on environmental, agronomic factors, plant parts
and the age (Topiar et al., 2015; Armando et al., 1997).
Major oral infectious diseases (dental caries and periodontal dis-
eases) are caused by bacteria colonizing the oral surfaces. Despite
the advances concerning its prevention and control, dental caries,
which is the result of the degradation of the enamel by the acid
produced by bacteria, is still considered a public health problem
worldwide affecting a large proportion of the young population
(Ishnava et al., 2013). The major causative bacteria of dental caries
are mutans group streptococci, such as Streptococcus mutans and
Streptococcus sobrinus (Berezow and Darveau, 2011; Selwitz et al.,
2007). Periodontal diseases are inflammatory disorders that lead
to tooth loss. They are caused by Gram-negative anaerobic bacteria
(Porphyromonas gingivalis, Fusobacterium nucleatum, and Aggregat-
ibacter actinomycetemcomitans) that destroy the periodontal tissue
by interacting with the mucosal immune cells (Allakerand Douglas,
2009; Madianos et al., 2005). Natural products have been recently
investigated as promising agents for the prevention of oral dis-
eases, and herbal products are increasingly used as alternatives to
traditional chemical drugs (Harzallah et al., 2011).
Few studies have reported the antioxidant activity of essen-
tial oils from E. globulus leaves (Mishra et al., 2010; Noumi et al.,
2011) and only one work has been conducted on their activity
against S. mutans (Goldbeck et al., 2014). In order to contribute
to the evaluation of the biological activities of E. globulus plant,
it should be important to test the antioxidant and the antibacte-
rial capacities of its leaves, with a view of their pharmaceutical
and industrial applications. This paper is a part of a study on the
evaluation of the volatile components of E. globulus cultivated in
Algeria, so the composition and the determination of two biolog-
ical activities (antioxidant and antibacterial) of the essential oils
of its leaves is the subject of this report. Therefore, in this work a
GC/MS method was developed to characterize the volatile com-
pounds from hydrodistillated extract of E. globulus leaves. Their
antioxidant effect was tested by the reducing capacity, the inhibi-
tion of lipid peroxidation and the scavenging effect on DPPH• free
radical. Concerning their antibacterial activity, it was determined
against Gram-negative periodontopathogenic and Gram-positive
cariogenic bacterial species.
2. Materials and methods
2.1. Plant materials and chemicals
Plant samples were collected from the arboretum of Derguinah
(36◦ 31 13.56 N, 5◦ 17 18.43 E), Bejaia, in the north east of Algeria,
in February 2013. All solvents and reagents were of analytical grade.
Samples were cleaned and dried in the drying oven at 30 ◦ C. A sam-
ple of 150 g boorishly crushed leaves was subjected to extraction
by hydrodistillation for 3 h/500 mL distilled water using a Clevenger
type apparatus. The obtained oil was recovered and stored at 4 ◦ C.
The oil yield was calculated as the ratio of the weight of oil to the
weight of leaves.
2.2. Determination of refractive index
The refractive index is used to confirm the purity of essential
oils. It was determined as previously described by Boukhatem et al.
(2014) and calculated using the Eq. (1).
Speed of light in a vacuum
n=
Speed of light in medium
149
2.3. Determination of specific gravity
The specific gravity of E. globulus oils was determined as previ-
ously described in AOAC (2000) standard method. Briefly, a gravity
bottle was weighted (W0 ), then filled with water and stopper was
inserted. The water of the bottle was wiped off and weighed again
(W1 ). The same process was repeated by using oil sample and
reweighted (W2 ). The specific gravity of the oils was calculated
using the Eq. (2).
W2 − W0
Specific gravity of oil = (2)
W1 − W0
Where W0 = weight of the empty gravity bottle, W1 = weight of
water + gravity bottle, W2 = weight of oil + gravity bottle.
2.4. Analysis of the essential oils
Analysis of the essential oils was carried out with a TRACE Ultra
Gas Chromatograph coupled to an ISQ Mass Spectrometer (Ther-
moScientific, Austin, Texas, USA), connected to a computer running
Xcalibur 2.0 software (ThermoScientific, Austin, Texas, USA). A
DB-5ms capillary column (60 m × 0.25 mm i.d., 0.25 ␮m film thick-
ness, Agilent J&W, Santa Clara, CA, USA) was used. The analysis
was performed using helium (purity >99.99 vol.%) as a carrier gas
at 1.2 mL/min with the following temperature program: 40 ◦ C for
2 min, increased to 250 ◦ C at 5 ◦ C/min and to 300 ◦ C at 30 ◦ C/min
and maintained at this temperature for 10 min. One ␮L of sample
was injected at a constant temperature of 250 ◦ C with a split ratio of
1:20 during 1 min. Masses were scanned between 40 and 650 uma.
The essential components were identified by comparing their mass
spectra with those stored in the NIST/EPA/NIH library.
2.5. Antioxidant activity
The antioxidant activity of the essential oils from E. globulus
leaves was estimated by DPPH, reducing power, and inhibition of
lipid peroxidation tests. The DPPH assay was estimated as described
by Noumi et al. (2011) method. Different concentrations of the sam-
ple were prepared in pure methanol, then 1 mL of each of them was
added to 0.25 mL of a 0.2 mmol/L DPPH methanolic solution (v/v).
The obtained solutions were shaken vigorously and left at room
temperature for 30 min, and their absorbance was measured at
517 nm after 30 min. The scavenging activity was calculated using
the Eq. (3).
[(A0 − At ) × 100]
DPHH scavenging activity (%) = (3)
A0
Where A0 is the absorbance of the control after 30 min, and At is
the absorbance of the sample after 30 min. Results were expressed
as IC50 (mg/mL), it corresponds to the dose required to cause a 50%
inhibition. A lower IC50 value corresponds to a higher antioxidant
activity.
The reducing capacity of the tested oils was evaluated by the
procedure of Singh et al. (2012). One mL of different concentrations
(10, 20, 30, 40 and 50 mg/mL) was mixed with 1 mL of phosphate
buffer (0.2 M ‘w/v’, pH 6.6) and 1 mL of potassium ferricyanide
[K3 Fe (CN)6 ], 1% ‘w/v’. The obtained solutions were incubated at
50 ◦ C for 20 min. Then 1 mL of Trichloroacetic acid (TCA) (10%‘w/v’)
was added to the solution that was then centrifuged for 10 min at
3000 × g. The supernatant was recovered and mixed with 1.5 mL
of distilled water and 150 ␮L of FeCl3 (0.1% ‘w/v’). The absorbance
was measured at 700 nm and the Butylated hydroxyanisole (BHA)
was used as standard. The result was expressed as IC50 (mg/mL).
The lipid peroxidation activity was determined by the ␤-
carotene bleaching method (Tepe et al., 2006), which is based on
(1)
the inhibition of the products of linoleic acid oxidation (volatile
150 L. Harkat-Madouri et al. / Industrial Crops and Products 78 (2015) 148–153

organic compounds and the conjugated dienehydroperoxides). An Table 1

Chemical composition of E. globulus essential oil obtained by hydrodistillation (HD)
emulsion of ␤-carotene/linoleic acid was prepared by mixing 25 ␮L
compared to the results of Topiar et al. (2015) * obtained by hydrodistillation (HDa )
of linoleic acid, 200 mg of Tween 40, 0.5 mg of ␤-carotene and 1 mL and supercritical fluid extraction (SFE).
of chloroform. After evaporation of the solvent, under low pres-
sure at 40 ◦ C, 100 mL of distilled water were added. To 2.5 mL of the Compounds Type KI Composition (%)

obtained solution, were added 350 ␮L of sample (2 mg/mL), after HD HDa* SFE*
shaking, the mixture was incubated for 48 h at room temperature. ˛-pinene M 920 4.61 – –
Two controls were prepared, one with the standard BHA (positive ˇ-pinene M 1122 0.07 9.25 3.50
control) and the other without BHA or extract (blank). Absorbance o-Ocymene M 1026 1.83 – –
at 490 nm of each sample was immediately measured at 0 h, 2 h, 4 h, Total M M 6.51 – –
Isovaleraldehyde OM 660 10.04 – –
13 h, and 48 h. Relative antioxidant activity was calculated accord- 2-pentanone-4-hydroxy-4- methyl OM 837 1.69 – –
ing to the Eq. (4). 1,8-Cineole OM 1033 55.29 36.68 21.01
Linalool OM 1096 0.10 – –
At
Antioxidant activity (%) = × 100 (4) 2-pinen-4-ol OM 1145 0.07 – –
A0 4-Terpineol OM 1181 0.70 – –
L-Pinocarvone OM 1162 0.10 – –
Where At is the absorbance of the sample after 48 h, and A0 is
˛-Terpineol OM 1196 5.46 – –
the essential oil absorbance at the beginning of incubation. Results Crypton OM 1189 3.10 – –
were expressed as IC50 (mg/mL). Cuminal OM 1243 0.42 – –
E-Neral OM 1268 0.63 – –
Phelandral OM 1279 0.10 – –
2.6. Antibacterial activity
Piperitone OM 1255 0.25 – –
p-cymenol OM 1297 0.45 – –
The antibacterial activity of essential oils from E. globulus Total OM 78.58 – –
leaves was tested against 12 bacterial strains. Six Gram-negative TOTAL (M + OM) 85.09 – –
periodontopathogenic bacteria (F. nucleatum ATCC 25586, A. acti- Aromadendrene S 1462 0.02 6.33 5.30
Allo-Aromadendrene S 1440 0.04 1.45 1.06
nomycetemcomitans ATCC 29522, P. gingivalis ATCC 33277, ATCC
Ledene S 1490 0.28 – –
49417, HW24D1, and W83) and six Gram-positive cariogenic bac- Total S 0.34 – –
teria (S. mutans ATCC 35668, ATCC 33535, ATCC 25175, S. sobrinus ␤ Caryophyllene-oxide OS 1551 0.14 – –
ATCC 33478, ATCC 27607, ATCC 27352). Epiglobulol OS 1563 0.21 1.00 0.32
Spathulenol OS 1577 7.44 – –
The growth of bacteria was carried out in Todd Hewitt broth
Caryophyllene-oxide OS 1586 1.66 – –
(BBL Microbiology Systems, Cockeysville, MA, USA) in the presence Globulol OS 1589 2.96 5.11 1.23
of 0.001% hemin and 0.0001% vitamin K (THB-HK). After 24 h of Eudesmol OS 1626 0.98 0.92 0.47
incubation at 37 ◦ C in an anaerobic chamber (N2 :H2 :CO2 ;75:10:15), Total OS 13.39 8.77 2.75
minimum inhibitory concentration (MIC) values were deter- TOTAL (S + OS) 98.82 – –

mined using a broth microdilution method in 96-well microplates
(Azelmat et al., 2015). A 24 h culture bacterium was prepared in
fresh THB-HK to obtain an optical density of 0.2 at 660 nm. Then
100 ␮L of both bacterium solution and serial dilutions of the essen-
tial oils, in culture medium containing 0.5% Tween 80, were mixed
into wells. Two controls were prepared (with no bacteria or no
essential oil), after that the microplates were incubated under
anaerobic conditions for 24 h at 37 ◦ C.
2.7. Statistical analysis
All tests were conducted in triplicate and results are expressed
as mean ± standard error. IC50 value was calculated using the lin-
ear regression equation obtained from the curve, i.e. absorbance = f
(extract concentrations). XLSTAT Release 10 (Addinsoft, Paris,
France) was used to the analysis of variance (ANOVA). To compare
means of each parameter, Tukey’s multiple range test (HSD) was
used. Differences were considered to be significant at P < 0.05.
Concentration (%): the percentage of concentrations based on peak area integration.
HD: hydrodistillation with 500 mL of water for 3 h.
HDa : hydrodistillation of Topiar et al. (2015) with 300 mL of water for 5 h.
SFE: CO2 supercritical fluid extraction from Topiar et al. (2015) at best operation
conditions of 12 MPa and 40 ◦ C and flow rate of 1.8 g/min.
KI: compounds were tentatively identified by comparison with mass spectra data
(MS) obtained from NIST/EPA/NIH library and confirmed by comparison with Kovat’s
index on DB5MS column.
M: monoterpenes.
S: sesquiterpenes.
OM: oxygenated monoterpenes.
OS: oxygenated sesquiterpenes.
*
Topiar et al. (2015).
2004). Furthermore, the values of these two physical properties
were in agreement with the AFNOR standards for E. globulus spices
(1.4590–1.4670 for the refractive index, and 0.906–0.923 for the
relative density) (AFNOR, 2000).

3. Results and discussion 3.2. Extraction yield of essential oils

3.1. Physical parameters analysis
Two physical parameters were determined to assess the quality
of oils of E. globulus leaves (refractive index and specific gravity).
The refractive index was 1.4657 ± 0.0070, which was compara-
ble to that previously reported in the literature (1.4602–1.46933)
(Boukhatem et al., 2014; Subramanian et al., 2012; Zrira et al.,
2004). Whereas, the relative density of the studied essential oils
was 0.9135 ± 0.0036. The farmer result was closer to those reported
in a previous work about the essential oils from Algerian E. glob-
ulus plant (0.919) (Boukhatem et al., 2014) and those reported
for the Moroccan Eucalyptus species (0.918–0.919) (Zrira et al.,
The volatile oils extracted from of E. globulus leaves were pale
colored, having camphor like smell and pleasant odor, similar to
the finding of Boukhatem et al. (2014) and Iqbal et al. (2003). The
extraction yield of essential oils was 2.53 ± 0.1%, this value is con-
siderably higher than those reported in previous studies, ranging
from 0.77% to 1.29% (da Silva et al., 2006; Joshi, 2012; Selvakumar
et al., 2012). Furthermore, it was higher than that obtained from
other species considered as economically important for essential
oils production, such as E. uniflora with 0.4–1.1% (Melo et al., 2007),
Psidium guajava with 0.13–0.45% (Joseph and Priya, 2010; Nisha
et al., 2011) and Melaleuca alternifolia with 1–2% yields (Carson
et al., 2006). Indeed, E. globulus plant also presents economic poten-
 L. Harkat-Madouri et al. / Industrial Crops and Products 78 (2015) 148–153 151

70 Table 2
Antioxidative capacities of the essential oil of E. globulus leaves and BHA.
60
Samples IC 50 (mg/mL)
Scavenging effect (%)

50 Reducing power DPPH ␤-carotene/linoleic acid

Essential oil 115.39 ± 1.45b 33.33 ± 0.55b 6.753 ± 0.39b

40
BHA 0.048 ± 0.015a 0.033 ± 0.002a 0.455 ± 0.19a

30 All the values are mean ± SD; SD: standard deviation.

a
Column wise values with different superscripts of this type indicate significant
20 difference (p < 0.05).
b
Column wise values with different superscripts of this type indicate significant
10 difference (p < 0.05).

0
0 10 20 30 40 50 60 ulus leaves. Besides 1,8Cineole, relative content of sesquiterpenes
Concentration (mg/mL) (S) and Oxygenated sesquiterpenes (OS) in E. globulus leaves essen-
tial oil extracted by the HD techniques (present study and Topiar’s
Fig. 1. Free radical scavenging activity (%) of the essential oil of E. globulus leaves.
work, 2015) and SFE was 13.93, 8.75% and 2.77%, respectively. This
indicates that HD remains the better technique for obtaining higher
tial, given that three to five thousand tonnes of Eucalyptus oils are yields of sesquiterpenes and oxygenated sesquiterpenes. In addi-
traded every year on international markets (Batish et al., 2008). tion, the HD used in the current study yielded more compounds (26
molecules) compared to the number of compounds (18 molecules)
3.3. GC/MS analysis of essentials oils obtained by HD and SFE from Topiar’s work (2015).

26 components have been identified in essential oils of of E. glob-
ulus leaves, which represent 98.82% of the total composition. They
are mainly composed of monoterpenes (85.09%); their detailed
composition is presented in Table 1. They consisted mostly of oxy-
genated monoterpenes and sesquiterpenes (78.58% and 13.39%,
respectively), 1,8-Cineole (55.29%), Spathulenol (7.44%) and ␣-
Terpineol (5.46%) being the main components (Table 1); similar
amounts of the predominant compound (1,8-Cineole) have been
reported in the literature (51.08 and 53.7%) (Boukhatem et al., 2014;
Elaissi et al., 2012). These contents depend essentially on environ-
mental, agronomic, age and geoclimatic factors and also on the used
extraction techniques and the experimental extraction conditions.
Table 1 lists the different compounds identified in the essential oils
of E. globulus leaves obtained by hydrodistillation (HD), compared
to those of Topiar et al. (2015) obtained by HD or supercritical fluid
extraction (SFE). The difference in the essential oils extracted by HD
and SFE is on the concentration of their components. In this study
the content of the major compound (1,8-Cineole) is higher (55.29%)
than those obtained by Topiar et al. (2015), which were about
36.68% and 21.01% for HD and SFE, respectively. Therefore, the HD
extraction is the best method to extract 1,8-Cineole from E. glob-
3.4. Antioxidant activity
Fig. 1 shows the result of the scavenging effect on DPPH radical
of the essential oils from E. globulus leaves. As we can see, the DPPH
scavenging capacity of the tested oils increased by increasing the
amount of sample, the inhibition percentage ranged from 11.72%
to 60.63% according to the tested concentrations. Mishra et al.
(2010) have reported a percentage of 79.55 ± 0.82 of leaf essential
oils (80% ‘v/v’ concentration) from Indian E. globulus. The activ-
ity of the tested oils (Table 2) is lower (IC50 = 33.33 ± 055 mg/mL)
than that of the standard BHA (IC50 = 0.033 ± 0.002 mg/mL). This
result differs from values previously reported for the commercial-
ized essential oils of the Tunisian E. globulus leaves with an IC50 of
57 ␮g/mL (Noumi et al., 2011), and that reported for the hydrodis-
tillated essential oils from the Indian E. citriodora with an IC50 of
425.4 ± 6.79 ␮g/mL (Singh et al., 2012).
Concerning the reducing power activity, results are shown
in Fig. 2, it increased with increasing concentrations. The IC50
values are depicted in Table 2, value of the standard BHA
(0.048 ± 0.015 mg/mL) is significantly lower than that of the tested
oils (115.39 ± 1.45 mg/mL). This activity is weak compared to that

0.6
y = 0.0028 x + 0.1808
R² = 0.9899
0.5
Absorbance at 700 nm (a.u)

0.4

0.3

0.2

0.1

0.0
0 20 40 60 80 100 120 140 160
Concentration (mg/mL)

Fig. 2. Reducing power of the essential oil of E. globulus leaves.

152 L. Harkat-Madouri et al. / Industrial Crops and Products 78 (2015) 148–153

Table 3
Minimum inhibitory concentration (MIC) of the essential oil of E. globulus leaves.

Gram-negative bacteria MIC (mg/mL) Gram-positive bacteria MIC (mg/mL)

F. nucleatum ATCC 25586 1.14 S. mutans ATCC 35668 11.4

A. actinomycetemcomitans ATCC 29522 9.13 S. mutans ATCC 33535 11.4
P. gingivalis ATCC33277 0.28 S. mutans ATCC 25175 11.4
P. gingivalis ATCC49417 4.56 S. sobrinus ATCC 33478 11.4
P. gingivalis HW24D1 2.28 S. sobrinus ATCC 27607 11.4
P. gingivalis W83 2.28 S. sobrinus ATCC 27352 11.4

reported in the literature (48 ␮g/mL) (Noumi et al., 2011), this dif- 0.400
ference may be explained by the fact that these researchers used Leaves BHA Control

Absorbance at 490 nm (a.u)

commercial essential oils rather than natural ones. On the other
hand, an IC50 value of 87.3 ± 9.27 ␮g/mL has been reported for the
essential oils of E. citriodora leaves (Singh et al., 2012), this value
being less important than that found in our study.
The inhibition of the lipid peroxidation activity was assayed by
the ␤-carotene bleaching test (Fig. 3), the activity of the oils was
found to be dose dependent. Its IC50 value (6.75 ± 0.39 mg/mL)
was significantly (p < 0.05) (Table 2) higher than that of BHA
(0.455 ± 0.19 mg/mL). This value is also higher than that of the
commercialized essential oils from E. globulus leaves (0.048 mg/mL)
(Noumi et al., 2011). Compared to the DPPH scavenging effect and
the reducing power, the essential oil of E. globulus leaves is more
active on the inhibition of the lipid peroxidation, presumably due
to the high specificity of the test for lypophilic compounds.
The difference found between our results and those reported in
other studies about the antioxidant activity of essential oils from
E. globulus plant, can be due to the difference in the mechanisms
involved in the assays applied to evaluate the different tests and the
extraction methods. The low activity of the tested oils can also be
explained by the abundance of the ineffective compounds. Indeed,
the tested oil is rich in monohydroxylated compounds such as 1,8-
Cineole, which is not able to chelate ferrous ions (AidiWannes et al.,
2010). AidiWannes et al. (2010) and MĎzami et al. (2013) have
reported that essential oils with higher monoterpenic compounds
are ineffective. Terpenes such as ␣-pinene, ␤-pinene, limonene,
␤-myrcene, sabinene and terpinolene are known to have a good
antioxidant properties, however, depending on the mechanism
involved in their action, some of them can exhibit low antiox-
idant activities (Martins et al., 2014). It has been reported that
the ␤-carotene bleaching ability of monoterpene hydrocarbons
may be due to the presence of methylene groups in their struc-
ture (AidiWannes et al., 2010). Consequently, the obtained results
suggest that ␤-carotene antioxidant capacity of the oils may be
lowered by its richness on monoterpene compounds. In the other
hand, a recent study has confirmed the weak antioxidant activity
of 1,8Cineole.
The low activity of the essential oil of E. globulus can also be
explained by the degradation of the bioactive compounds during
their extraction by hydrodistillation. Indeed, the hydrodistillation
can lead to the thermal degradation, hydrolysis, and solubilisation
of the bioactive compounds in water, thus changing their antiox-
idant capacity. Furthermore, the water used in hydrodistillation
makes several antioxidants unstable or degrades them by enzy-
matic action in the wet plant material. In fact, in hydrodistillation,
samples were usually extracted in boiling water over a long period
of time, which could lead to thermal decomposition of thermo-
labile target compounds from E. globulus and thus lowering the
antioxidant activity of the extract (Bagheri et al., 2014).
3.5. Antibacterial activity
Natural antibacterial substances, including essential oils, can
be incorporated into mouthwash to control dental plaque. More
specifically, ListerineTM which has been widely used for many
0.350
0.300
0.250
0.200
0.150
0.100
0.050
0.000
0 10 20 30 40 50 60
Time (hours)
Fig. 3. Antioxidant activity of the essential oil of E. globulus leaves, BHA and control,
measured by ␤-carotene/linoleic acid test.
years, contains thymol, eucalyptol, menthol and methyl salicylate
(Allaker and Douglas, 2009).
As depicted in Table 3, Gram-negative bacteria were more sensi-
tive to the essential oil of E. globulus, with the MIC ranged from 0.28
to 9.13 mg/mL. P. gingivalis ATCC 33277 was the most susceptible
(MIC = 0.28 mg/mL) followed by F.nucleatum (MIC = 1.14 mg/mL). ;
Goldbeck et al. (2014) have reported an MIC of 0.013 mg/mL for S.
mutans, which is much lower than that found in our study, which
was about 11.4 mg/mL for all tested strains of S. mutans. The diver-
gence of our results and those of Goldbeck et al. (2014) can be
related to the difference in the composition of the tested essential
oils. Indeed, these two essential oils depict significant differences
in their 1,8-Cineole and ␣-pinene concentrations (55.29% vs 71.05%
and 8.30% vs 4.61%, respectively). Moreover, these two compounds
were known by their antibacterial activity (Chikhoune et al., 2013;
Elaissi et al., 2012). The sensitivity of P. gingivalis ATCC33277 bac-
teria (MIC = 0.28 mg/mL) to the essential oils of E. globulus, may be
due to the presence of the oxygenated monoterpene compounds
(␣-Terpineol, MIC = 0.4 mg/mL) (Park et al., 2012).
Essential oils are slightly more active against Gram-positive
than Gram-negative microorganisms, this can be explained by the
presence of an outer membrane around their cell wall, which
can limit the diffusion of hydrophobic compounds through its
lipopolysaccharide covering. However, the sensitivity of Gram-
positive bacteria to the essential oils has been reported (Wilkinson
et al., 2003). Essential oils of Mentha piperita have shown a greater
activity against S. enteritidis than against L. monocytogenes, when
added to the Greek appetizers. In the other hand, no differences
have been detected between the sensibilities of Gram-positives and
Gram-negatives after 24 h. But, the inhibition effect was more pro-
nounced with Gram-negative than with Gram-positive organisms
after 48 h (Burt, 2004).
4. Conclusion
This study reports the antibacterial effect of the essential oil
from E. globulus leaves against periodontopathogenic bacterial
 L. Harkat-Madouri et al. / Industrial Crops and Products 78 (2015) 148–153
species. This essential oil is found to be more active against
Gram-negative bacteria, with weak antioxidant activity. The chem-
ical identification of the different molecules characterizing the
E. globulus essential oil evidenced the presence of oxygenated
monoterpenes which can act as antibacterial agents. Essential oil of
E. globulus leaves, being a significant antibacterial compounds, may
thus have a potential application for pharmaceutical formulation,
such as toothpaste and mouthwash.
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Further reading
Sharopov, F.S., Wink, M., Setzer, W.N., 2015. Radical scavenging
and antioxidant activities of essential oil components—an experi-
mental and computational investigation. Nat. Prod. Commun. 10,
153–156.